Neuroblastoma, treated with a variety of chemotherapeutic drugs, frequently responds, but drug-resistant tumors generally return. To explore these drug resistance mechanisms we isolated and biochemically characterized mutant clones of a spontaneous murine C-1300 neuroblastoma in monolayer cell culture resistant to antifolates, anthracyclines and microtubule disrupters. Maytansine-vincristine-adriamycin-Bakers antifol reciprocally cross-resistant cells, resistant to structurally and functionally diverse drugs, are unable to accumulate these drugs despite normal binding to cell-free homogenates. This, lowered tumorigenicty in the syngeneic Ajax mouse and collateral sensitivity to glysosylation inhibitors led us to examine the resistant cell's plasma membrane glycoproteins and glycolipids using whole cell metabolic or surface radiolabeling techniques and fractionation by 2-dimensional gel electrophoresis or thin layer chromatography. All mutants demonstrate specific but pleiotropic changes in cell-surface glycoproteins and gangliosides suggesting a common glycoconjugate altering enzyme. Double-minute chromosomes, seen in unstable but not more highly resistant stable drug-resistant descendents, may contain amplified gene copies of the resistance mediating protein. One glycoprotein, X, MW 100,000, pI equals 4.0, missing from several mutants and present in a revertant, may be causal. We propose to determine whether this or other altered glycoconjugates represent a glycosyl-transferase, glycosidase, protein synthesis or membrane assembly defect, to contrast this mechanism with that of diploid human neuroblastoma SK-SY-5Y drug-resistant clones, and to compare the effect of the membrane glycosylation inhibitor, tunicamycin, on drug-resistance and altered tumorigenesis in an effort to better understand and improve tumor chemotherapy.